Fibrinolytic and thrombolytic enzymes have been extensively used in the treatment of thromboembolic vascular diseases, with remarkable clinical results. The high thrombolytic power of fibrinolytic enzymes are incorporated in anti-thrombic materials which have such enzymes immbolized on the surfaces of polymeric materials, and because of their excellent anti-thrombic activities, such materials have been found to be very useful as a variety of blood-contact materials (see, for example, Igaku no Ayumi (Progress of Medical Science), vol. 101, p. 144, 1971).
It has however turned out that the activities of the immobilized fibrinolytic enzymes decrease more or less with time, as in the case of unimmobilized enzymes. This limits the utility of the anti-thrombic materials in spite of their high activities because, for one thing, they cannot be stored for an extended period at room temperature, and for another, they have to be stored under special refrigerated conditions in order to prevent activity drop.
Further, anti-thrombic materials having fibrinolytic enzymes immobilized on polymeric surfaces must be sterilized before they are used in medical fields. However, enzymes are instable physiological substances and their activities can be reduced or entirely lost as a result of sterilization. The deactivation of enzymes is therefore another serious problem that must be solved before they are used in anti-thrombic materials.
Many proposals have been made with a view to enhancing the storage stability of the activity of unimmobilized fibrinolytic enzymes, and they include the use of protamine, as described in Japanese Patent Application (OPI) No. 117486/1977 (The term "OPI" as used herein refers to a "published unexamined Japanese Patent Application".); chlorhexidine or salts thereof, as described in Japanese Patent Application (OPI) No. 120186/1977; dextran sulfate and salts thereof, as described in Japanese Patent Application (OPI) Nos. 89083/1979, 34082/1980, and 145591/1977; albumin, as described in Japanese Patent Application (OPI) No. 142592/1978; sulfate esters of sugars, as described in Japanese Patent Application (OPI) No. 32691/1979; glycoproteins, such as mucin and mucoid, as described in Japanese Patent Application (OPI) No. 44004/1979; guanine and guanine derivatives, as described in Japanese Patent Application (OPI) No. 46885/1979; gelatin, as described in Japanese Patent Application (OPI) Nos. 64688/1979 and 80406/1979; chondroitin sulfate, as described in Japanese Patent Application (OPI) No. 67089/1979; hydroxyethyl starch, dextran, and nicotinamide, as described in Japanese Patent Application (OPI) No. 70419/1979; hydrodextrin sulfate, as described in Japanese Patent Application (OPI) No. 68290/1980; hydroxypropyl cellulose, as described in Japanese Patent Application (OPI) No. 23891/1981; and a compound having at least one of disulfido group and mercapto group, as described in Japanese Patent Application (OPI) No. 37882/1979.
Use of amino acids has also been proposed, such as in Japanese Patent Application (OPI) No. 142593/1978 (amino acids, sugars and neutral salts), Japanese Patent Application (OPI) No. 18486/1973 (neutral amino acids), Japanese Patent Application (OPI) No. 80284/1974 (amines, amides and ammonium ion), and Japanese Patent Application (OPI) No. 147916/1979 (human serum albumin and polar amino acids). In three of these patents, amino acids are shown to be used in combination with other compounds. In Japanese Patent Application (OPI) No. 142593/1978, glycine, lysine and arginine are mentioned as amino acids; in Japanese Patent Application (OPI) No. 80284/1974 (corresponding to U.S. Pat. No. 3,950,223), arginine (amino acid) is mentioned as an amine; and in Japanese Patent Application (OPI) No. 147916/1979 (corresponding to U.S. Pat. No. 4,244,943), polar amino acids are used together with human serum albumin, including arginine, aspartic acid, glutamic acid, histidine, lysine, serine and threonine, among which lysine, arginine and histidine are classified as basic amino acids.
As shown above, there exist a host of patents disclosing methods for enhancing the storage activities of unimmobilized fibrinolytic enzymes, but no method has been described in connection with immobilized fibrinolytic enzymes. As for the elimination or reduction of the enzymatic activity resulting from sterilization, unimmobilized fibrinolytic enzymes are generally used without sterilization, so there is no wonder that the literature is completely devoid of proposals for preventing the activities of such enzymes from being eliminated or reduced as a result of sterilization. However, immobilized fibrinolytic enzymes must be sterilized before they are used in medical fields, but no technique has been successfully developed so far in order to prevent the inactivation of such immobilized enzymes resulting from sterilization.